Project Summary In eukaryotes, cellular DNAs are normally sequestered either in the nucleus or in mitochondria. However, if DNA, such as that derived from viruses, enters the cytoplasm, it is sensed by cGAS, the cyclic GMP-AMP (cGAMP) synthase, a critical component of the innate immune system. cGAMP, generated from ATP and GTP, upon DNA-dependent activation of cGAS, binds STING, which promotes interferon generation and the inflammatory response. However, it is unknown, how cGAS responds to mitotic chromosomes, when they are exposed to the cytoplasm after nuclear envelope break down at the onset of mitosis. The long-term goal of this project is to understand how cGAS and the innate immunity system respond to mitotic failures. Our unpublished data show that cGAS does not bind to nuclear DNA during interphase, but does interact with chromosomal DNA during mitosis. Furthermore, cGAS also accumulates on DNA in micronuclei, which are generated through chromosome missegregation and have compromised nuclear envelope integrity. We hypothesize that cGAS affects the fate of cells undergoing aberrant mitosis, such as those induced by treatment with the microtubule-stabilization drug, taxol (paclitaxel). Taxol is commonly used for cancer chemotherapy, but the mechanistic basis for its efficacy remains unclear. Taxol treatment inhibits proper spindle assembly and chromosome segregation, and may lead to extended mitotic delay. Cells arrested in mitosis are known to take one of two fates; cell death in mitosis and slippage into interphase without proper chromosome segregation. The mechanism by which these fates are chosen has not been established. Based on our preliminary data we hypothesize that cGAS does not respond to chromosomal DNAs during normal progression of mitosis, but promotes cell death when cells are arrested in mitosis for an extended period. We also hypothesize that cGAS accumulating in micronuclei generated after mitotic failure induces inflammatory responses and/or autophagy. In this grant, we aim to 1) establish the role of cGAS in promoting mitotic cell death, 2) to dissect the mechanism by which cGAS is activated during mitosis, 3) to delineate the pathway that acts downstream of cGAS to promote mitotic cell death, and 4) to understand the consequence of cGAS binding to DNA in micronuclei. To achieve our goal, we will combine cutting edge techniques, including genome engineering, live microscopy, and biochemical reconstitutions. The outcomes of this study will define a novel paradigm for how aberrant mitosis is sensed by the innate immune system, and will have broad implications for the molecular mechanisms behind cGAS function, as well as for the medical application of anti- mitotic drugs.